Figure 4.

Gene modification strategies for use in induced pluripotent stem cell (iPSC) lines. Three basic strategies can be employed for gene editing. To create single allele homozygous expression (hemizygous) lines, Module A targets a single allele of the gene of interest (GOI) in iPSC lines that are heterozygous for the risk variant. Targeting efficiency for hemizygous clones is approximately 20% of green fluorescent protein positive puromycin resistant clones, and allele targeting is random so that either the protective or risk allele can be modified. Module B generates complete knockout of the GOI on a background of homozygous protective alleles. Module C utilizes GOI-knockout lines to re-express either the protective or risk variant of the GOI using a CRISPR/Cas9 platform that targets integration into the adeno-associated virus integration site 1 (AAVS1) on chromosome 19.